This study protocol conforms to the Guide for the Care and Use of Laboratory Animals [National Institutes of Health, (NIH) Bethesda, MD, USA] and was approved by the Institutional Animal Care and Use Committee of the Nanjing Medical University for Laboratory Animal Medicine (IACUC-2005043).
Human umbilical MSCs (purchased from the Clinical Center of Reproductive Medicine of Nanjing Medical University) were cultured in α-minimal essential medium (α-MEM) containing 10% fetal bovine serum (FBS). MSCs were exposed to control or 50 ng/ml IFN-γ (300-02, Peprotech, USA) conditions for 48 hours. H9c2 and human umbilical vein endothelial cells (HUVECs) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS. All media and reagents used for cell culture were purchased from Gibco (Carlsbad, USA). For normal culture, cells were incubated at 37°C, 21% O2, and 5% CO2. Under oxygen and glucose deprivation conditions, cells were cultured under 1% O2, 5% CO2, 94% N2 and serum deprivation conditions.
Exosomes Extraction and Characterization
MSCs (1×106) were cultured to 70% confluence and then treated with 6ml of Exosome-free FBS (Gibco) for 2d. The cell culture medium (6ml) was collected in a 15ml centrifuge tube and centrifuged at 1,500 × g for 30 min to remove the cells and debris. Transferred the processed supernatant to another 15ml centrifuge tube containing 2ml RiboTM exosome extraction reagent (for cell culture medium, C10130-2, Ribobio, China). These mixtures were incubated overnight at 4°C, and then centrifuged at 2,000 ×g for 30 min. The supernatant was discarded, and the exosomal pellet was resuspended in 100μl PBS.
The exosomal surface markers were detected using western blotting with anti-TSG101, CD63, and CD81 antibodies (Abcam, UK). The external electron microscope (TEM) was utilized to observe the appearance of Exos. The Exos were fixed with 1% glutaraldehyde, and then coated with 1% phosphotungstic acid on a copper mesh. JEM-2100 transmission electron microscope (JEOL, Tokyo, Japan) was used to observe the sample. Nanoparticle tracking analysis (NTA) was applied to analyze the size and distribution of Exos. We recorded and tracked the Brownian motion of Exos in PBS (Carlsbad, California). The particle size distribution data was obtained with Stoke-U.S. A ZetaView PMX 110 system (Particle Metrix, Germany) was used for NTA.
Exosomes uptake assay
In order to demonstrate the uptake of Exos by H9c2 cells and HUVEC cells, Exos were labeled with Dil (red fluorescent dye, C1036, Beyotime, China) and co-cultured with recipient cells at 37°C for 6h or 24h and then washed with PBS and fix with 4% paraformaldehyde for 20 min. The nuclei were stained with 6-diamino-2-phenylindole (DAPI) (0.5g/ml, Beyotime) for 10 min, and observed with a confocal microscope.
Flow cytometry (KeyGEN Biotech, China) detected cell apoptosis. H9c2 cells were seeded in 1×105/6-well tissue culture plates overnight, and treated with different Exos or PBS before OGD. The cells were washed with PBS and stained with Annexin V fluorescein isothiocyanate and propidium iodide apoptosis kit (KeyGen Biotech, China). Flowjo Software version 10.0 (Tree Star, USA) was used to analyze apoptotic cells. TdT-mediated dUTP Gap End Labeling (TUNEL) Apoptosis Detection Kit (Roche, USA) was also used to detect cell or tissue apoptosis according to the manufacturer's instructions. The formula for calculating the percentage of apoptotic nuclei was the total number of TUNEL stained nuclei divided by the total number of TUNEL positive nuclei.
Cultured HUVECs in a 6-well plate, scraped the confluent layer with the tip of a P200 pipette. Then washed and incubated the cells after adding 100 µg/well of different Exos. Images were taken before and 6h, 12h after incubation, and Image J software (NIH) was used to determine the reduction ratio of the scratch area.
Tube Formation Assay
HUVECs were treated with PBS or different Exos. Then cells were washed with PBS and seeded (30,000 cells/well) in 96 well plates coated with growth factor reduced Matrigel (Corning, United States). After 6h, capillary-like tube formation was observed and photographed. Tube length and number of branches were analyzed with Image J software (NIH).
Quantitative real-time PCR (qRT-PCR)
The total cellular and exosomal RNA was extracted using Trizol reagent (Life Technologies, USA) according to the manufacturer’s instructions.14 A stem-loop-specific primer method was used to measure miR-21-5p expression, as described previously.20 The sequences of primers used in the study were shown in Table S1. The relative expression was calculated using the following equation: relative gene expression = 2− (ΔCtsample − ΔCtcontrol). All samples were measured in triplicate.
Transfection of miR-21-5p mimics (50 nmol/L) and negative control miRNA (50 -100 nmol/L) synthesized by Guangzhou Ribobio were carried out into H9c2 cells using riboFECTTM CP Reagent (Ribobio, China) according to the instructions of manufacturer. The full-length BTG2 sequence and empty vector as negative control were inserted into a pcDNA 3.1 plasmid (GenePharma, China) to transfect H9c2 cells using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer instructions. STAT1 siRNA and Negative control FAM manufactured by Suzhou GenePharma were delivered into MSCs by Lipofectamine 2000. qRT-PCR were performed to determine transfection efficacy. At 48h after transfection, different groups of cells were harvested for further study.
Protein extraction and Western blot (WB) analysis were performed as previously stated.14 Briefly, cells were washed with PBS and lysed with lysis buffer on ice for 20 minutes. The total cell protein concentration was detected by the BCA Protein Assay Kit. The total protein (20μg) was separated using SDS-PAGE (Invitrogen) and transferred to a PVDF membrane (Roche). The membrane was blocked with 5% bovine serum albumin (0.1%) in TBS-Tween and incubated against the desired antibody. The primary antibodies used for Western blot analysis are listed in Table S2. Bands were visualized using enhanced chemiluminescence reagents and analyzed using a gel documentation system (Bio-Rad Gel Doc1000 and Multi-Analyst version 1.1).
MI Model, Histological Analysis, and Immunofluorescence Staining
Eight-week-old male Sprague-Dawley (SD) rats obtained from the experimental animal center of Nanjing Medical University were randomly divided into 4 groups: sham operation group (Sham group, n=6), PBS injection group (PBS group, n=6), Ctrl-Exo injection group (Ctrl-Exo group, n=6), and IFN-γ-Exo injection group (IFN-γ-Exo group, n=6). Briefly, as previously described 14, left anterior descending artery (LAD) was ligated, and Exos (50µL, 1µg/µL) or PBS was injected around the infarct area in rats. All surgeries and subsequent analyses were blinded for intervention. Echocardiography (Vevo 3100) was performed to determine the left ventricular ejection fraction (LVEF) and left ventricular short axis shortening rate (LVFS) after 2w and 4w.
The rats were sacrificed 4w after surgery. Inflammatory cell infiltration and cell arrangement was evaluated by Hematoxylin-Eosin (HE) staining. Masson's trichrome staining was used to evaluate fibrosis and collagen area after MI, CD31 immunofluorescence staining was to observe the distribution of microvessels. Apoptosis was detected by TUNEL staining (Roche, USA). The primary antibody was anti-CD31 (ab7388; British abcam). DAPI was used for nuclear counterstaining. The images were further analyzed using a fluorescence microscope (Zeiss, Germany) and Image J software (NIH).
Continuous variables and categorical variables were described as mean ± SD and percentages, respectively. Independent-Sample T-test was used to compare continuous variables between the two groups. One way Analysis of variance (ANOVA) followed by Tukey’s correction was used for comparison of three or more groups. All statistical tests were performed using GraphPad Prism software version 8.0, and p < 0.05 was considered statistically significant.